Introducing Potential Key Proteins and Pathways in Human Laryngeal Cancer: A System Biology Approach

The most common malignant neoplasm of the head and neck region is laryngeal cancer which presents a significant international health problem. The present study aims to screen potential proteins related to laryngeal cancer by network analysis to further understanding disease pathogenesis and biomarker discovery. Differentially expressed proteins were extracted from literatures of laryngeal cancer that compare proteome profiling of patient›s tissue with healthy controls. The PPI network analyzed for up and down regulated proteins with Cytoscape Version 3.4. After PPI construction, topological properties of the two networks have been analyzed. Besides, by using MCODE. the Gene Ontology (GO) analysis, the related modules and pathways were examined. Our study screened 275 differentially changed proteins, including 136 up- and 139 down-regulated proteins. For each network, it has been considered 20 key proteins as hub and 20 as bottleneck. A number of 26 hub-bottleneck nodes is introduced for the two networks. A total of 11 modules including 6 downregulated and 5 upregulated network modules were obtained. The most significant GO function in the significant upregulated module was the RNA processing, and the most significant one in the downregulated module with highest score was the respiratory electron transport chain. Among 275 investigated proteins, 12 crucial proteins are determined that 4 of them can be introduce as a possible biomarker panel including YWHAZ, PPP2R1A, HSP90AA1, and CALM3 for human laryngeal cancer.


Introduction
The most common malignant neoplasm of the head and neck regions is laryngeal cancer which presents a significant international health problem.This type of cancer has high rate of mortality because of the poor diagnosis in early stage of the disease.Despite favorable treatment in early-stage laryngeal cancers, survival rates for advanced-stage disease are less than 50%.Surgery and chemotherapy are two suitable treatment options that are used for laryngeal cancer.However, their combination is also is used.Recently the number of patients treated with radiotherapy and chemotherapy is increased (1) .However, survival is decreased (2).Laryngeal cancer has been considered as a multifactorial disease associated with the interaction between environmental factors and genetic background (3).Environmental factors of laryngeal cancer are introduced as a lower consumption of vegetables and fruits, and higher consumption of milk, eggs, meat, tea, alcohol, and smoking (4).Recently, various studies have established the changes in molecular level which are associated with the development of laryngeal cancer.For example, several studies have investigated associations between CYP1A polymorphisms and laryngeal cancer risk (5).Alcohol consumption or smoking beside the uridine diphosphate glucuronosyl transferase enzyme (UGTs) rs4148323 act synergistically to increase the risk of laryngeal cancer (6).It has also reported the relationship between this type of cancer and nucleotide excision repair pathway genes such as ERCCs and XPA (7).The proteomics studies on laryngeal cancer show that the changed expression proteins regulate cellular proliferation, differentiation, and apoptosis that may directly related to the pathogenesis of cancer (8).Another one reported that some significantly changed expression proteins were the products of oncogenes and others were related to signal transduction and immune defense (9).Deeb A and colleagues showed that related DNA repair pathways are curtail in larynx cancer patients (10).For better understanding of molecular mechanisms of laryngeal cancer pathogenesis, protein-protein interaction (PPI) network analysis can provide an informative concept and detail schema (11)(12)(13)(14)(15)(16)(17)(18)(19)(20).Therefore, we used a systems biology approach (based on the available proteomics literature data) as a rational strategy to reveal novel specific markers and probably therapeutic targets for laryngeal cancer.

Data collection
In this study, the inclusion criteria were the studies on the human species using cell line and laryngeal squamous tissue samples involved in the comparison between the tumor and normal tissues.Exclusion criteria were the studies on non-human tissue and studies on samples of biological fluids, including plasma, serum, saliva, and urine.Studies only involved in comparison between the tumor tissue and tumor metastasis one.There was no limitation in methods in proteomic studies.We manually evaluated the publications in line with the above conditions; a total of 275 significantly changed expression proteinsextracted of which 136 proteins belong to up regulated protein group and 139 proteins were as down regulated proteins (See Tables 1  and 2).

PPI network analysis
PPI network analyzed by Cytoscape Version 3.4 and Betweenness centrality (BC) and node degree the two major centrality parameters were analyzed by using a Cytoscape plug-in called 'Network Analyzer' (21).Degree indicates the number of connectivity belongs to a node and nodes having high degree were introduced as hub proteins.BC value the other centrality index reflects the shortest paths that pass through a node (22).

Screening of network modules and functional analysis
The modules of the two constructed networks (including up and down regulated networks) were provided by MCODE analysis and parameters including Node Score Cutoff: 0.2, K-Core: 2, Degree Cutoff: 2 and, Max depth = 100 were used as the cut-off criteria for network module screening.MCODE score > 3 and node > 6 were considered for functional enrichment analysis of the modules.Kappa statistic ≥ 0.4 and Bonferroni step down method for probability value correction were used for annotation analysis of the selected modules.

Results
After the submission of up-regulated and down-regulated proteins into Cytoscape, a total of 7312 and 6707 nodes related to the up-regulated and down-regulated proteins are included in the networks, respectively.In the final networks (Figures 1 and 2), the node›s degree was organized based on size; the nodes with high degree have bigger size and the blue to brown color represented low to high BC values for each node.\ The nodes with high degree were considered as key proteins.Then, the top 20 proteins with highest connectivity were identified as the hub proteins for each of the networks and similarly, the top 20 proteins based on betweenness centrality value were selected as bottleneck proteins (See Tables 3 and 4).

Module analysis
A total of 11 modules including 5 up-regulated and 6 down-regulated network modules were obtained using default criteria.It was selected modules with MCODE score > 3 and node > 6.Five up-regulated modules (Up, 1-5) (Figure 3), and six down-regulated modules (Down, 1-6) (Figure 4) were selected for enrichment analysis.
There were some key proteins (hubs) in total of 5 up-regulated modules and 3 up-regulated network modules among them have 3 seed proteins (see Table 5).While, in down-regulated network modules, only Down-1 module has seed.The hubs in this network are distributed as tabulated data in Table 5.

Functional enrichment analysis for modules
Four up-regulated modules (Up, 1-4) and three down-regulated modules (Down, 1-3) were enriched based on functional annotation.The top three GO terms for each module are shown in Table 6.

Discussion
Protein-protein interaction (PPI) network analysis has a significant growth in cancer studies to facilitate introducing early stage biomarkers (23).In our study, the laryngeal cancer related proteins were analyzed via PPI network construction, hub gene identification, module analysis, and functional enrichment analysis of most significant modules.These stages were carried out for up-regulated proteins and down-regulated ones in human laryngeal cancer tissue, separately.As it is shown in Tables 1 and 2, there are 275 changed expression proteins (including up and down regulated proteins) related to the human tissue of laryngeal cancer.Data management and analysis is a difficult process due to huge numbers of the collected proteins.Since PPI network analysis considered as key proteins.Then, the top 20 proteins with highest connectivity were identified as the hub proteins for each of the networks and similarly, the top 20 proteins based on betweenness centrality value were selected as bottleneck proteins (See Tables 3 and 4).  is a powerful method in categorization and ranking of the candidate and related proteins for a certain disease, here the up and down regulated networks are constructed separately (Figures 1  and 2).Topological analysis of the networks lead to rank of the nodes based on networks properties (18).By using two centrality indices including degree and betweenness, totally 80 nodes are selected among 275 initial proteins as important proteins (see Tables 3 and 4).However, the number of 80 nodes can not be considered as a suitable biomarker panel related to laryngeal cancer and more screening is required.The hub-bottleneck nodes for the up and down regulated networks are shown in  3 and 4 and Table 5 there are 5 and 6 modules for the up and down regulated networks respectively.Functional enrichment analysis for top score modules indicated that RNA processing and splicing, mitotic cell cycle regulation and sugar biosynthesis are affected by up-regulated modules while metabolic pathways and mitochondria are the main affected subjects     by down regulated modules (see Table 6).The most significant pathways in four modules Up, 1-4 were RNA processing, G1/S transition mitotic cell cycle, protein ubiquitination and RNA splicing.It has been revealed overlapping between important pathways involved in the conversion of pre-mRNA to mature mRNA.In previous studies, it shows that polymorphisms of mRNA processing genes can be considered as risk factors for development of laryngeal cancer (25).The most significant pathways in down regulated modules (Down, 1-3) were respiratory electron transport chain, regulation of protein insertion in to mitochondrial membrane   There were some key proteins (hubs) in total of 5 up-regulated modules and 3 up-regulated network modules among them have 3 seed proteins (see Table 5).While, in down-regulated network modules, only Down-1 module has seed.The hubs in this network are distributed as tabulated data in Table 5.According the results of Table 5, the scattering of hubs in up-modules was more than down ones.Interestingly, the finding indicate that the seeds and hubs in up-modules have the similar functions with each other that are associated with regulation of cell cycle (29, 30).Among 26 hubbottleneck nodes 12 proteins (8 up-regulated and 4 down-regulated proteins) are distributed in 8 modules (see Table 5).These proteins are tabulated in supplementary Table S1 and are ranked based on amounts of degree value.Here two suggestions are feasible: first investigation about expression changes of these 12 genes in the field and the second idea is selection of the top up and down regulated genes for more examinations.We choose cutoff 1200 for degree and therefore YWHAZ and PPP2R1A as the top two up-regulated genes and also HSP90AA1 and CALM3 as the top two down-regulated genes are introduced as human laryngeal cancer.YWHAZ gene with the highest degree and BC scores encodes 14-3-3 protein zeta/delta that has an essential role in tumor cell proliferation (31) through the regulation of multiple cellular processes, such as cell cycle control, antiapoptosis, signal transduction, inflammation, and cell adhesion/motility (32).YWHAZ has been introduced as candidate proto-oncogene in head and neck squamous cell carcinoma whose reduced expression causes lower level of DNA synthesis rates (33).14-3-3 proteins could be a key regulatory components in many processes that are crucial for development of cancers (34) such as laryngeal cancer (8).PPP2R1A gene encodes one subunit of protein phosphatase 2. This protein phosphatase is involved in control of cell growth and cell division processes.The role of this subunit in integrity of enzyme is highlighted.Therefore, it is expected that PPP2R1A plays a crucial regulatory role in cell proliferation in cancer cell line(35).HSP90AA1 and CALM3 were found as two top ranked genes in the down-regulated PPI network.These proteins belong to family of proteins which involved in the regulation of specific target proteins in cell cycle control and programmed cell death (36, 37).On the other hand, CALMs in addition to cell cycle, related to centrosome cycle and deregulation of this protein can be the origin of chromosomal instability in cancer (38).Interestingly, all determined possible biomarkers are related to the cell cycle process.

Conclusion
In this study, it has been represented a model of important proteins and pathways that provide a new level of information for laryngeal cancer

Figure 1 .
Figure 1.Protein-protein interaction network for up-regulated differentially expressed proteins in tissue of human laryngeal cancer include of 7312 nodes and 33757 edges.

Figure 1 .
Figure 1.Protein-protein interaction network for up-regulated differentially expressed proteins in tissue of human laryngeal cancer include of 7312 nodes and 33757 edges.

Figure 1 .
Figure 1.Protein-protein interaction network for up-regulated differentially expressed proteins in tissue of human laryngeal cancer include of 7312 nodes and 33757 edges.

Figure 2 .
Figure 2. Up: Centrality analysis of protein-protein interaction network for down-regulated differentially expressed proteins in tissue of human laryngeal cancer consist of 6707 nodes and 27422 edges.Down: The dense and central part of upper network is shown in more details.

Figure 2 .
Figure 2. Up: Centrality analysis of protein-protein interaction network for down-regulated differentially expressed proteins in tissue of human laryngeal cancer consist of 6707 nodes and 27422 edges.Down: The dense and central part of upper network is shown in more details.

Figure 3 .
Figure 3. Modules of the protein-protein interaction network for up-regulated differentially expressed proteins (MCODE score > 3 and node > 6).The yellow cycles indicate seed proteins and the pink cycles reagent proteins in modules.There are no seed in Up-4 and Up-5 modules.

Figure 3 .
Figure 3. Modules of the protein-protein interaction network for up-regulated differentially expressed proteins (MCODE score > 3 and node > 6).The yellow cycles indicate seed proteins and the pink cycles reagent proteins in modules.There are no seed in Up-4 and Up-5 modules.

Figure 4 .
Figure 4. Modules of the protein-protein interaction network for down-regulated differentially expressed proteins (MCODE score > 3 and node > 6).The yellow cycles indicate seed proteins and the pink cycles reagent proteins in modules.Only Down -1 module has seed and the other ones have no seed.

Figure 4 .
Figure 4. Modules of the protein-protein interaction network for down-regulated differentially expressed proteins (MCODE score > 3 and node > 6).The yellow cycles indicate seed proteins and the pink cycles reagent proteins in modules.Only Down -1 module has seed and the other ones have no seed.

Table 1 .
The list of up-regulated genes in tissue of human laryngeal cancer.

Table 2 .
The list of down-regulated genes in tissue of human laryngeal cancer.

Table 3 .
Presentation of the hub proteins in the up-regulated and down-regulated protein-protein interaction networks of laryngeal cancer (top 20 in each PPI network).The hub nodes that play as bottleneck node are asterisked (for more details see Table4 and discussion).

Table 4 .
The list of top 20 up-regulated and down-regulated genes ranked based on BC from largest to smallest values.

Table 3
. As it is shown in this Table there are 15 and 11 hubbottlenecks for up and down regulated networks respectively.Module is a part of a network including closed related proteins havig specific biological function (20).Determined modules of network can provide informative perspective about different roles of the nodes (24).As it is shown in Figures

Table 3 .
Presentation of the hub proteins in the up-regulated and down-regulated protein-protein interaction networks of laryngeal cancer (top 20 in each PPI network).The hub nodes that play as bottleneck node are asterisked (for more details see Table4 and discussion).

Table 4 .
The list of top 20 up-regulated and down-regulated genes ranked based on BC from largest to smallest values.

Table 5 .
The modules of up regulated and down regulated PPI networks of human tissue of laryngeal cancer.The asterisked proteins are hub-bottleneck nodes.

Table 6 .
GO functional enrichment analysis of up-regulated and down-regulated PPI network modules.Top three terms of each module are tabulated.